The long-range goals of the proposed research are to elucidate the molecular and cellular mechanisms by which 17-estradiol (E2) regulates hypothalamic kisspeptin (Kiss1) neuronal circuits in females. Kiss1 neurons are a major target of E2 and are essential for pubertal development and adult reproduction. These neurons are also important for sensing and relaying gonadal status to neurons vital for regulation of energy balance, including the hypothalamic arcuate proopiomelanocortin (POMC) and neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons. In spite of the importance of Kiss1 neurons, relatively little is known about the biophysical properties and molecular signature of these cells, as well as the neural circuits by which they operate. We have discovered that the Kiss1 neurons in the rostral periventricular area of the third ventricle (RP3V) express the critical ion channels and receptors that permit spontaneous E2-dependent rebound burst firing. Based on the E2-dependent burst firing characteristics and their projections to arcuate neurons, we propose the novel hypothesis that the RP3V Kiss1 neurons serve as the central pacemaker neurons that provide E2-dependent excitatory drive to the anorexigenic POMC neurons and inhibitory drive to the orexigenic NPY/AgRP neurons. This kisspeptin input to POMC and NPY/AgRP neurons is vital for the estrogenic control of energy homeostasis during the ovulatory cycle in females. Elucidating the cell-specific signaling pathways and gene expression at the single cell level will help in developing new strategies for targeting hormone actions in CNS neurons. Our multidisciplinary approach incorporates a unique set of cellular, molecular, optogenetic and chemogenetic tools and our combined expertise in molecular biology, electrophysiology, histochemistry and whole animal physiology to address the following aims: 1) To elucidate the sodium channel (Nav) subunit composition and the contribution of INaP to the generation of burst firing in RP3V Kiss1 neurons in high- versus low-E2 states. 2) To elucidate the postsynaptic actions of kisspeptin on arcuate POMC and NPY/AgRP neurons using single cell RT-PCR and whole-cell recording. 3) To elucidate the direct synaptic input to arcuate POMC and NPY/AgRP neurons from RP3V Kiss1 neurons using optogenetic stimulation and whole-cell recording. 4) To examine the in vivo effects of selective chemogenetic activation or inhibition of RP3V Kiss1 neurons on food intake in low- versus high-E2 females, respectively. Collectively, these experiments will allow for the elucidation of the ion channels important for the pacemaker activity in hypothalamic Kiss1 neurons, and how this translates into actions within hypothalamic neurocircuits that are vital for not only energy homeostasis but other autonomic functions.